One of the most important mechanisms in formation of embryonic nervous systems is the guidance of axons and growth cones by directional guidance cues (Goodman, Annu. Rev. Neurosci. 19 (1996), 341-77; Mueller, Annu. Rev. Neurosci 22, (1999), 351-88). A suitable model system for studying this guidance process is the retinotectal system of vertebrates. In the chick embryo approximately 2 million retinal ganglion cell (RGC) axons leave each eye and grow towards the contralateral tectum opticum to form a precise map (Mey & Thanos, (1992); J. Hirnforschung 33,673-702). Having arrived at the anterior pole of the optic tectum, RGC axons start to invade their tectal target to find their target neurons. Mapping occurs in such a way that RGC axons from nasal retina project to posterior tectum and temporal axons to anterior tectum. Along the dorso-ventral axis, axons coming from dorsal retina terminate in ventral tectum, whereas those from ventral retina end up in dorsal tectum. Ultimately, a precise topographic map is formed, where neighborhood relationships in the retina are preserved in the tectum so that axons from neighboring ganglion cells in the retina synapse with neighboring tectal neurons. Most important for formation of this map are graded tectal guidance cues, read by retinal growth cones carrying corresponding receptors which also show a graded distribution (Sperry, Proc. Natl. Acad. Sci. USA 50 (1963), 703710; Bonhoeffer & Gierer, Trends Neurosci. 7 (1984) 378-381).
Position of each retinal growth cone in the tectal field is therefore determined by two sets of gradients: receptor gradients on in-growing retinal axons and growth cones and ligand gradients on tectal cells (Gierer, Development 101 (1987), 479-489). The existence of the graded tectal ligands has been postulated from anatomical work. Their identification, however, proved to be extremely difficult and was only made possible with the development of simple in vitro systems (Walter ; Development 101 (1987), 685-96; Cox, Neuron 4 (1990), 31-7). In the stripe assay, RGC axons grow on a membrane carpet, consisting of alternating lanes of anterior (a) and posterior (p) tectal membranes. On these carpets, temporal retinal axons grow on anterior tectal membranes and are repelled by the posterior lanes, whereas nasal axons do not distinguish between a and p membranes (Walter, Development 101 (1987), 685-96). The same specificity is also observed in the growth cone collapse assay (Raper & Kapfhammer, Neuron 4 (1990), 21-29) where temporal retinal growth cones collapse after addition of posterior tectal membrane vesicles but do not react to anterior tectal vesicles and where nasal growth cones are insensitive to either type of vesicles (Cox, (1990), loc. cit.). In both assay systems, treatment of posterior tectal membranes with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC) (which cleaves the lipid anchor of glycosylphosphatidylinositol (GPI)-linked proteins) removed their repellent and collapse-inducing activity (Walter, J. Physiol 84 (1990), 104-10).
One of the first repulsive guidance molecules identified in the retinotectal system of chick embryos was a GPI-anchored glycoprotein with a molecular weight of 33/35 kDa (Stahl, Neuron 5 (1990), 735-43). This 33/35 kDa molecule, later termed RGM (Repulsive Guidance Molecule), was active in both stripe and collapse-assays and was shown to be expressed in a low-anterior high-posterior gradient in the embryonic tecta of chick and rat (Mueller, Curr. Biol. 6 (1996), 1497-502; Mueller, Japan Scientific Societies Press (1997), 215-229). Due to the abnormal biochemical behavior of RGM, the precise amino acid sequence was not easily obtainable. RGM was described as a molecule which is active during vertebrate development. Interestingly, RGM is downregulated in the embryonic chick tectum after E12 and in the embryonic rat tectum after P2 and completely disappears after the embryonic stages (Muller (1992), Ph. D thesis University of Tübbingen; Müller (1997) Japan Scientific Societies, 215-229). In 1996, Müller (loc. cit.) showed that CALI (chromophore-assisted laser inactivation) of RGM eliminates the repulsive guidance activity of posterior tectal membranes. However, due to the presence of other guidance molecules, in particular of RAGS (repulsive axon guidance signal) and ELF-1 (Eph ligand family 1), a complete elimination of guidance was not always detected and it was speculated that RGM acts in concert with RAGS (now termed ephrin-A5) and ELF-1 (ephrin-A2). It was furthermore envisaged that RGM may be a co-factor potentiating the activity of RAGS and ELF-1 in embryonic guidance events.
In 1980/81 the group of Aguayo found that, when peripheral neurons are transplanted/grafted into injured CNS of adult, axon growth of CNS neurons is induced (David, Science 214 (1981), 931-933). Therefore, it was speculated that CNS neurons have still the ability and capacity of neurite-outgrowth and/or regeneration, if a suitable environment would be provided. Furthermore, it was speculated that “CNS-neuron regeneration inhibitors” may exist.
In 1988, Caroni and Schwab (Neuron 1,85-96) described two inhibitors of 35 kDa and 250 kDa, isolated from rat CNS myelin (NI-35 and NI-250; see also Schnell, Nature 343 (1990) 269-272; Caroni, J. Cell Biol. 106 (1988), 1291-1288).
In 2000, the DNA encoding for NI-220/250 was deduced and the corresponding potent inhibitor of neurite growth was termed Nogo-A (Chen, Nature 403 (2000), 434-438). The membrane-bound Nogo turned out to be a member of the reticulon family (GrandPre, Nature 403 (2000), 439-444).
Further factors which mediate neuronal outgrowth inhibition have first been isolated in grasshoppers, and termed “fasciclin IV” and later “collapsin” in chicken. These inhibitors belong to the so-called semaphorin family. Semaphorins have been reported in a wide range of species and described as transmembrane proteins (see, inter alia, Kolodkin Cell 75 (1993) 1389-99, Püschel, Neuron 14 (1995), 941-948). Yet, it was also shown that not all semaphorins have inhibitory activity. Some members of the family, e.g. semaphorin E, act as an attractive guidance signal for cortical axons (Bagnard, Development 125 (1998), 5043-5053).
A further system of repulsive guidance molecules is the ephrin-Eph system. Ephrins are ligands of the Eph receptor kinases and are implicated as positional labels that may guide the development of neural topographic maps Flanagan, Ann. Rev. Neurosc. 21 (1998), 309-345). Ephrins are grouped in two classes, the A-ephrins which are linked to the membrane by a glycosylphosphatidylinositol-anchor (GPIanchor) and the B-ephrins carrying a transmembrane domain (Eph nomenclature committee 1997). Two members of the A-ephrins, ephrin-A2 and ephrin-A5, expressed in low anterior-high posterior gradients in the optic tectum, have recently been shown to be involved in repulsive guidance of retinal ganglion cell axons in vitro and in vivo (see, inter alia (Drescher, Cell 82 (1995), 359-70; Cheng, Cell 79 (1994), 157-168; Feldheim, Neuron 21 (1998), 563-74; Feldheim, Neuron 25 (2000), 563-74). Considering the fact that a plurality of physiological disorders or injuries are related to altered cellular migration processes, the technical problems underlying the present invention was to provide for means and methods for modifying developmental or cellular (migration) processes which lead to disease conditions.
The Ephrin, Semaphorin, Slit, and RGM families of extracellular guidance cues specify axonal trajectories during nervous system development1-3. The netrins are a family of proteins that are profound modulators of growth of developing axons, functioning as attractants for some axons and repellents of other axons. As such, the modulation of these effects provides an important therapeutic pathway for assisting the regeneration of axons in adult nervous system (e.g. following injury or trauma). While neuronal receptors have been identified for most axonal guidance cues, the mechanism by which the recently sequenced RGM protein (WO 02/051438) acts has not been clarified3. As described in part above, chick RGM is expressed in a posterior to anterior tectal gradient and has been shown to collapse temporal but not nasal retinal growth cones3. After signal peptide cleavage and GPI addition, the cell surface RGM is proteolytically processed to a mature active form of 33 kDa3.
The ability to construct high-throughput and specific pharmaceutical screens for modulators of guidance cues (such as RGM) has been limited by the lack of identifiable receptors. Identifying receptors on axons that mediate neural responsiveness to guidance cues will provide key targets for identifying lead pharmaceuticals for therapeutic intervention in the nervous system (see, for example, U.S. Pat. Nos. 6,087,326 and 5,747,262). Accordingly, because RGM has a demonstrated role in axon growth, it would be desirable to accurately identify the receptor through which RGM acts such that targeted screens could be conducted.
Neogenin is known to share sequence similarity with the Netrin receptor Deleted in Colorectal Cancer (DCC). The sequence for the Neogenin gene has been described (for example, Keeling S L, Gad J M, Cooper H M. “Mouse Neogenin, a DCC-like molecule, has four splice variants and is expressed widely in the adult mouse and during embryogenesis.” Oncogene. Aug. 7, 1997;15(6):691-700. GenBank NT—039474; NM—008684) and it has been previously theorized that it is an interaction with Netrin-1 that is responsible for signaling through Neogenin. However, as described in detail herein, the present inventors have determined the true physiological ligand for Neogenin.